The identification of effective chemotherapy and appropriate surgical treatment for patients with osteosarcoma has led to significant improvements in outcome. The determination of prognosis in osteosarcoma continues to be based on clinical staging systems. The identification of additional prognostic factors would allow stratification of therapy, which may improve the durability of "high risk" patients and minimize toxicity to the "good risk" patients. Investigation of biological features related to chemotherapy response may identify prognostic factors. Advances in the understanding of the molecular basis of resistance to methotrexate, an agent routinely used in high doses for the treatment of this disease has emerged, allowing the development of in vitro assays which may predict response to this drug. Studies of these biological factors may identify new antifolate inhibitors of dihydrofolate reductase which overcome the relevant mechanisms of resistance as being potentially more effective for the treatment of osteosarcoma. The purpose of this study is to investigate biological factors related to methotrexate resistance as predictors of histologic response to preoperative chemotherapy and outcome in patients with osteosarcoma. Osteosarcoma tumor samples obtained from patients treated by the Childrens' Cancer Group and the Pediatric Oncology Group will be assayed for alterations in methotrexate uptake, methotrexate polyglutamylation as well as reduced folate carrier, dihydrofolate reductase, folylpolyglutamate synthestase and gamma- glutamyl hydrolase expression. The results of these assays will be correlated with the histologic response of the tumors to preoperative chemotherapy and patient survival to potentially identify these assays as prognostic factors. These studies may define the incidence of intrinsic and acquired methotrexate resistance in tumor samples and suggest the use of new antifolates and therapeutic strategies in the treatment of osteosarcoma.